B25716/1: a novel albumin-binding Gd-AAZTA MRI contrast agent with improved properties in tumor imaging.

The aim of this study is to describe the synthesis of, relaxometric characterization of, pharmacokinetic properties of, and animal imaging experiments with a new, low molecular weight gadolinium complex with high binding affinity toward serum albumin. The gadolinium(III) chelate (B25716/1) is based on the structure of the heptadentate ligand 1,4-bis(hydroxycarbonylmethyl)-6-[bis(hydroxycarbonylmethyl)]amino-6 methylperhydro-1,4-diazepine (AAZTA) covalently conjugated to an analogue of deoxycholic acid. The study was conducted as a comparison with that of an analogous complex based on the octadentate diethylenetriaminepentaacetic acid ligand B22956/1 (whose albumin binding properties were previously assessed). The structural modification with respect to B22956/1 leads to a system that can host two coordinated water molecules in fast exchange with bulk water with potential higher efficiency as an MRI contrast agent. On interaction with human serum albumin the expected-field-independent-relaxation enhancement is not observed, possibly as a consequence of the displacement of one of the two inner-sphere water molecules of the gadolinium complex. At clinically relevant magnetic fields, however, the plasma relaxivity of B25716/1 is markedly higher than that shown by B22956/1, owing to concomitant synergistic contributions from the electronic correlation time and water molecules in the second coordination sphere. The capability of B25716/1 to enhance tumor regions in magnetic resonance images was assessed in vivo at 3 T on a xenograft tumor mouse model prepared with PC-3 cells. B25716/1 displays signal enhancements approximately double those observed for B22956/1, in agreement with the findings of the in vitro relaxivity investigations.

In vivo and in vitro liver cancer metabolism observed with hyperpolarized [5-(13)C]glutamine.

Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized (13)C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-(13)C]glutamine, which provides a highly sensitive (13)C MR marker. With this preparation of hyperpolarized [5-(13)C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-(13)C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.

Development and validation of a smoothing-splines-based correction method for improving the analysis of CEST-MR images.

Chemical exchange saturation transfer (CEST) imaging is an emerging MRI technique relying on the use of endogenous or exogenous molecules containing exchangeable proton pools. The heterogeneity of the water resonance frequency offset plays a key role in the occurrence of artifacts in CEST-MR images. To limit this drawback, a new smoothing-splines-based method for fitting and correcting Z-spectra in order to compensate for low signal-to-noise ratio (SNR) without any a priori model was developed. Global and local voxel-by-voxel Z-spectra were interpolated by smoothing splines with smoothing terms aimed at suppressing noise. Thus, a map of the water frequency offset ('zero' map) was used to correctly calculate the saturation transfer (ST) for each voxel. Simulations were performed to compare the method to polynomials and zero-only-corrected splines on the basis of SNR improvement. In vitro acquisitions of capillaries containing solutions of LIPOCEST agents at different concentrations were performed to experimentally validate the results from simulations. Additionally, ex vivo investigations of bovine muscle mass injected with LIPOCEST agents were performed as a function of increasing pulse power. The results from simulations and experiments highlighted the importance of a proper 'zero' correction (15% decrease of fictitious CEST signal in phantoms and ex vivo preparations) and proved the method to be more accurate compared with the previously published ones, often providing a SNR higher than 5 in different simulated and experimentally noisy conditions. In conclusion, the proposed method offers an accurate tool in CEST investigation.

Cellular labeling with Gd(III) chelates: only high thermodynamic stabilities prevent the cells acting as 'sponges' of Gd3+ ions.

MR-labeling of cells may be carried out by adding a Gd-based contrast agent to the incubation media. The amount of gadolinium internalized in HTC and C6 cells upon incubation with Gd-DTPA-BMA is circa one order of magnitude higher than those found with Gd-DTPA, Gd-DOTA and Gd-HPDO3A, respectively. The comparison of relaxometric and mass spectrometry determinations allows us to establish that only a minor fraction of intact Gd-DTPA-BMA is internalized into the cells. Moreover the binding/uptake behavior shown by Gd-DTPA-BMA resembles that found when GdCl(3) is added to the incubation medium. We suggest that the lower stability of Gd-DTPA-BMA is responsible for a shift in the dissociation equilibrium that results in the net transfer of Gd(3+) ions on the cell membrane followed by a slower internalization process. The transmetallation process is mediated by components of the incubation media, among which a dominant role is represented by phosphate anions. The uptake of Gd(3+) ions is clearly reflected in the drastic decrease of cell viability observed for cells labeled with Gd-DTPA-BMA.

Targeting cells with MR imaging probes based on paramagnetic Gd(III) chelates.

The low sensitivity is the major disadvantage of MRI as compared to PET. Therefore, amplification strategies are necessary for specific pathway labeling. This survey is aimed at exploring different routes to the entrapment of Gd(III) chelates in various type of cells at amounts sufficiently large to allow MRI visualization. Namely, the obtained results have been summarized in terms of internalization via i) pinocytosis; ii) phagocytosis; iii) receptors; iv) receptor mediated endocytosis; v) transporters; vi) transmembrane carrier peptides. MRI visualization of cells appears possible when the number of internalized Gd(III) chelates is of the order of 10(7)-10(8)/cell. Pinocytosis shows to be particularly useful for labeling cells that can be incubated for several hours in the presence of high concentrations of Gd-agent. This approach appears very effective for labeling stem cells. Nanoparticles filled with Gd-chelates can be used for an efficient loading of cells endowed with a good phagocytic activity. Entrapment via receptors most often results in receptor mediated endocytosis. Suitably functionalized monomeric and multimeric Gd-chelates can be considered for being internalized by this route as well as supramolecular systems such as those formed between Avidin and biotinylated Gd-complexes. Exploitation of up-regulated transporters of nutrients in tumor cells appears to be a promising route for their differentiation from healthy cells. Finally, properly designed systems entering the cells by means of penetrin-like peptides deserve great attention.

Agmatine induces apoptosis in rat hepatocyte cultures.

BACKGROUND/AIMS: Agmatine, the compound formed by decarboxylation of arginine, is believed to be an endogenous neurotransmitter through interaction with the imidazoline receptors. However, it also appears to regulate rat hepatocyte polyamines by modifying both their synthesis and their catabolism. As the decrease in polyamine content has been correlated with apoptosis, we examined the possibility that agmatine has an effect on this phenomenon. METHODS: Apoptotic cells were detected by visualizing nuclear shrinkage/fragmentation in hepatocytes cultured at 21 and 5% oxygen tension. Caspase-3 activity, cleavage of PARP, release of cytochrome c and mitochondrial swelling were therefore measured in the two conditions and in the presence or not of agmatine. RESULTS: In rat hepatocytes agmatine promoted apoptosis, procaspase 3 processing and increase of caspase-3 like activity. This occurred through mitochondria swelling and release of cytochrome c. Cyclosporin A and catalase blocked the swelling. CONCLUSIONS: Our experiments show that agmatine, besides all the known biological effects, has also part, at least in hepatocytes, in the modulation of programmed cell death.

Radiochemical nitric oxide synthase activity determination in rat brain with fast and accurate HPLC analysis.

In the central nervous system, NOS activity is involved in several physiological events, such as refinement of afferent connections in development, or linking cerebral blood flow to neural activity in adulthood, and also in many pathological events, such as cell death in brain ischemia and regulation of vasospasm in hemorrhage. Therefore, we studied NOS activity in the CNS. We describe a fast and accurate method in which we use HPLC analysis to identify and quantify citrulline eluted by ion-exchange chromatography, thus implementing the current method to evaluate NOS activity. This technique could be readily applied for NOS activity determination not only in brain, but also in all other tissues.

Transport and metabolism of agmatine in rat hepatocyte cultures.

Rat hepatocytes in culture take up [14C]-agmatine by both a high-affinity transport system [KM = 0.03 mM; Vmax = 30 pmol x min x (mg protein)-1] and a low-affinity system. The high-affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4-guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C-4-aminobutyrate is also accumulated in the presence of an inhibitor of 4-aminobutyrate transaminase.

Long chain diamines inhibit growth of C6 glioma cells according to their hydrophobicity. An in vitro and molecular modeling study.

A series of diamines with the general structure NH2(CH2)xNH2, x=2-12, was tested for their potential effects on cell proliferation of cultured rat C6 glioma cells in comparison to natural polyamines. Long chain diamines reduced cell number after 48 h in culture with a sequence of 1,12-diaminododecane (1,12-DD) >1,10-diaminodecane >1,9-diaminononane. Polyamines (putrescine, spermidine and spermine) as well as diamines up to a CH2-chain length of x=8 were found to be ineffective. The spermine analogue 1,12-DD was the most effective molecule in reducing cell number in an irreversible, dose-dependent manner (EC50=3 microM under serum-free conditions). In further experiments we investigated the mechanisms of action of 1,12-DD. The compound had only a minor effect on cell cycle and did not affect free internal calcium concentration. Under physiological conditions 1,12-DD interacts with triplex DNA but not with duplex DNA. Ornithine decarboxylase activity as well as the concentration of internal polyamines were found to be reduced by 1,12-DD. Polyamine application, however, was not able to reverse the effect of 1,12-DD, indicating a polyamine-independent or non-competitive mechanism of action. 1,12-DD reduced cell number by induction of apoptosis as well as necrosis. In molecular modeling studies it was found that a minimal hydrophobic intersegment of at least 4 A was required to make a diamine an effective drug in respect to cellular growth. A hydrophobic gap of this size fits the minimum requirement expected from molecular modeling to provide space for hydrophobic interactions with parts of proteins like a CH3-group. Our results show that 1,12-DD acts as a potent drug, reducing the number of C6 glioma cells, and suggest that its spatial and hydrophobic properties are responsible for its mechanism of action.

Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase.

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.